The human gut harbors a dense and complex community of microbes known as the intestinal microbiota. A disrupted or dysbiotic microbial ecosystem has been connected to several diseases including inflammatory bowel diseases, metabolic syndrome, and colon cancer. Therapeutic manipulation of the gut microbiota has broad implications for human health. However, the abundance of bacterial strains that reside within an individual is largely stable over time and can be difficult to alter in a predictable, robust, and reproducible fashion. Fecal microbiota transplants are being increasingly used to repair a dysbiotic microbiota, however, the fate of the introduced strains is unpredictable and largely individualized (i.e., influenced by the recipient's microbiota). If microbiota reprogramming is to become an effective therapy for a broad range of diseases, a better understanding of the factors that control entrenchment of therapeutic strain integration and the role of diet in the reinforcement of new strains is critically important. In Aim 1, microbiota accessible carbohydrates (MACs), powerful levers on microbiota composition and function, will be used to elucidate the mechanisms by which exogenous bacterial species entrench within an established microbiota. Furthermore, new environmental niches will be established through novel dietary carbohydrates to replace pathogenic bacterial strains with commensal isogenic strains. State-of-the-art synthetic biology tools and imaging will be employed to quantify the biogeographical location of newly entrenching strains within the colon. In Aim 2, microbes and genetic loci will be isolated from the microbiomes of hunter-gatherer populations and cognate polysaccharides will be identified. Since the diet and carbohydrate utilization enzymes encoded within the microbiota of traditional populations is distinct from that of US residents, these pre-industrialized microbiomes possess a large collection of carbohydrate active enzymes not present or exceedingly rare in the Western world. Isolation and utilization of unique MACs will be tested to enable engraftment of therapeutic strains that have been engineered to utilize these carbohydrates. Whether using multiple privileged MACs is an effective strategy to entrench and control the abundance of multiple species independently within multiple established microbiotas will be tested in a humanized mouse model (mice harboring a human microbiota). This proposal establishes a new paradigm for reprogramming a dysbiotic microbiota, which makes possible the robust and reproducible entrenchment of exogenous bacterial strains. The ultimate goal of this strategy is to enable stable incorporation of new species and functions that could be tailored to an individual's specific microbiota and disease state.